Functional validation of mungbean LEA protein coding gene in bacterial expression system confers salt stress tolerance

Authors

  • Rajesh SUBRAMANIAN Tamil Nadu Agricultural University, Centre for Plant Molecular Biology and Biotechnology, Coimbatore 641 003, Tamil Nadu; Tamil Nadu Agricultural University, Horticultural College and Research Institute, Periyakulam 625 604, Tamil Nadu (IN)
  • Nandhini U. PANDI Tamil Nadu Agricultural University, Centre for Plant Molecular Biology and Biotechnology, Coimbatore 641 003, Tamil Nadu (IN)
  • Radhamani THANGAVEL Tamil Nadu Agricultural University, Centre for Plant Molecular Biology and Biotechnology, Coimbatore 641 003, Tamil Nadu (IN)
  • Likhith R.K. SWAMY Tamil Nadu Agricultural University, Centre for Plant Molecular Biology and Biotechnology, Coimbatore 641 003, Tamil Nadu (IN)
  • Srimathi Priya LAKSHMINARAYANAN Tamil Nadu Agricultural University, Horticultural College and Research Institute, Periyakulam 625 604, Tamil Nadu (IN)
  • Shenbagavalli SANTHAMANI Tamil Nadu Agricultural University, Horticultural College and Research Institute, Periyakulam 625 604, Tamil Nadu (IN)
  • Backiyavathy M. RAVALAN Tamil Nadu Agricultural University, Horticultural College and Research Institute, Periyakulam 625 604, Tamil Nadu (IN)
  • Rajangam JACOB Tamil Nadu Agricultural University, Horticultural College and Research Institute, Periyakulam 625 604, Tamil Nadu (IN)

DOI:

https://doi.org/10.15835/nbha52313416

Keywords:

characterization, E. coli, LEA, protein expression, salinity, plant stress

Abstract

Mungbean (Vigna radiata R. Wilczek) is a major tropical food grain legume that is widely cultivated in tropical part of the world. Mungbean like other plants, tolerate and survive limited water situation owing to expression of stress associated proteins that offers membrane stability and cell protection. Late Embryogenesis Abundant (LEA) proteins are among the group of low molecular weight proteins, that play diverse roles in stress protection in several species of plants and animals. A LEA protein coding gene VrLEA2 was isolated from mungbean and its role in stress tolerance has been demonstrated using a bacterial expression system. VrLEA2 gene isolated was of size 893 bp and characterized as a group 1 LEA protein based on the sequence signature motif with presence of hydrophilic domain and a characteristic 20-mer conserved amino acids motif. VrLEA2 gene was cloned into a bacterial expression vector, pET 28a (+), transformed into the E.coli BL21 (DE3) cells for recombinant protein expression and subsequently subjected to antibiotic selection with kanamycin. Functional validation of the VrLEA2 for salt stress tolerance with varied concentration of NaCl (0 mM to 600 mM) showed alteration in colony morphology and reduction in the number of colonies in control compared to the transformed cells demonstrating the improved survival rate of cells expressing VrLEA2 protein. These findings indicate the best use of bacterial expression system for functional validation of plant proteins under stressed environments. 

References

Ahn YJ, Jung M (2023). Improved recombinant protein production using heat shock proteins in Escherichia coli. Biocatalysis and Agricultural Biotechnology 20:102736. https://doi.org/10.1016/j.bcab.2023.102736

Almoguera C, Jordano J (1992). Developmental and environmental concurrent expression of sunflower dry-seed-stored low-molecular-weight heat-shock protein and Lea mRNAs. Plant Molecular Biology 19(5):781-792. https://doi.org/10.1007/BF00027074

Altunoglu YC, Baloglu MC, Baloglu P, Yer EN, Kara S (2017). Genome-wide identification and comparative expression analysis of LEA genes in watermelon and melon genomes. Physiology and Molecular Biology of Plants 23(1):5-21. https://doi.org/10.1007/s12298-016-0405-8

Baker J, Van dennSteele C, Dure L (1988). Sequence and characterization of 6 Lea proteins and their genes from cotton. Plant molecular biology 11(3):277-291. https://doi.org/10.1007/BF00027385

Bimboim H, Doly J (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Research 7(6):1513-1523. https://doi.org/10.1093/nar/7.6.1513

Boudet J, Buitink J, Hoekstra FA, Rogniaux H, Larré C, Satour P, Leprince O (2006). Comparative analysis of the heat stable proteome of radicles of Medicago truncatula seeds during germination identifies late embryogenesis abundant proteins associated with desiccation tolerance. Plant physiology 140(4):1418-1436. https://doi.org/10.1104/pp.105.074039

Chen J, Li N, Wang X, Meng X, Cui X, Chen Z, Ren H, Ma J, Liu H (2021). Late embryogenesis abundant (LEA) gene family in Salvia miltiorrhiza: identification, expression analysis, and response to drought stress. Plant Signaling and Behavior 16(5):1891769. https://doi.org/10.1080/15592324.2021.1891769

Colmenero-Flores JM, Campos F, Garciarrubio A, Covarrubias AA (1997). Characterization of Phaseolus vulgaris cDNA clones to water deficit: identification of a novel embryogenesis abundant- like protein. Molecular Biology 35:393-405. https://doi.org/10.1023/A:1005802505731

Dagert M, Ehrlich S (1979). Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells. Gene 6(1):23-28. https://doi.org/10.1016/0378-1119(79)90082-9

Dalal M, Tayal D, Chinnusamy V, Bansal KC (2009). Abiotic stress and ABA-inducible Group 4 LEA from Brassica napus plays a key role in salt and drought tolerance. Journal of Biotechnology 139(2):137-145. https://doi.org/10.1016/j.jbiotec.2008.09.014

Doyle JJ, Doyle JL (1990). Isolation of plant DNA from fresh tissue. Focus 12:13-15.

Dure L, Crouch M, Harada J, Ho TH, Mundy J, Quatrano R, Thomas T, Sung ZR (1989). Common amino acid sequence domains among the LEA proteins of higher plants. Plant Molecular Biology 12:475-486. https://doi.org/10.1007/BF00036962

Espelund M, Sæbøe‐Larssen S, Hughes DW, Galau GA, Larsen F, Jakobsen KS (1992). Late embryogenesis‐abundant genes encoding proteins with different numbers of hydrophilic repeats are regulated differentially by abscisic acid and osmotic stress. The Plant Journal 2(2):241-252. https://doi.org/10.1046/j.1365-313X.1992.t01-46-00999.x

Ganesan K, Xu B (2018). A critical review on phytochemical profile and health promoting effects of mung bean (Vigna radiata). Food Science and Human Wellness 7(1):11-33. https://doi.org/10.1016/j.fshw.2017.11.002

Gao J, Lan T (2016). Functional characterization of the late embryogenesis abundant (LEA) protein gene family from Pinus tabuliformis (Pinaceae) in Escherichia coli. Scientific Reports 6:19467. https://doi.org/10.1038/srep19467

Gao T, Mo Y, Huang H, Yu J, Wang Y, Wang W (2021). Heterologous expression of Camellia sinensis late embryogenesis abundant protein gene 1 (CsLEA1) confers cold stress tolerance in Escherichia coli and yeast. Horticultural Plant Journal 7(1):89-96. https://doi.org/10.1016/j.hpj.2020.09.005

Garay-Arroyo A, Colmenero-Flores JM, Garciarrubio A, Covarrubias AA (2000). Highly hydrophilic proteins in prokaryotes and eukaryotes are common during conditions of water deficit. Journal of Biological Chemistry 275(8):5668-5674. https://doi.org/10.1074/jbc.275.8.5668

Gaubier P, Raynal M, Hull G, Huestis GM, Grellet F, Arenas C, Delseny M (1993). Two different Em-like genes are expressed in Arabidopsisthaliana seeds during maturation. Molecular and General Genetics 238(3):409-418. https://doi.org/10.1007/BF00292000

He JX, Fu JR (1996). The research progresses in Lea proteins of seeds. Plant Physiology Communications 241-246.

Hong-Bo S, Zong-Suo L, Ming-An S (2005). LEA proteins in higher plants: structure, function, gene expression and regulation. Colloids and surfaces B: Biointerfaces 45(3-4):131-135. https://doi.org/10.1016/j.colsurfb.2005.07.017

Huang R, Xiao D, Wang X, Zhan J, Wang A, He L (2022). Genome-wide identification, evolutionary and expression analyses of LEA gene family in peanut (Arachis hypogaea L.). BMC Plant Biology 22(1):155. https://doi.org/10.1186/s12870-022-03462-7

Huwaidi A, Pathak N, Syahir A, Ikeno A (2018). Escherichia coli tolerance of ultraviolet radiation by in vivo expression of a short peptide designed from late embryogenesis abundant protein. Biochemical and biophysical research communications 503 (2):910-914. https://doi.org/10.1016/j.bbrc.2018.06.095

İbrahime M, Kibar U, Kazan K, Yüksel Özmen C, Mutaf F, Demirel Aşçı S, Çakır Aydemir B, Ergül A (2019). Genome-wide identification of the LEA protein gene family in grapevine (Vitis vinifera L.). Tree Genetics and Genomes 15:1-14. https://doi.org/10.1007/s11295-019-1364-3

Imai R, Chang L, Ohta A, Bray EA, Takagi M (1996). A lea-class gene of tomato confers salt and freezing tolerance when expressed in Saccharomyces cerevisiae. Gene 170 (2):243-248. https://doi.org/10.1016/0378-1119(95)00868-3

Juszczak I, Bartels D (2017). LEA gene expression, RNA stability and pigment accumulation in three closely related Linderniaceae species differing in desiccation tolerance. Plant Science 255:59-71. https://doi.org/10.1016/j.plantsci.2016.10.003

Kim HS, Lee JH, Kim JJ, Kim CH, Jun SS, Hong YN (2005). Molecular and functional characterization of CaLEA6, the gene for a hydrophobic LEA protein from Capsicum annuum. Gene 344:115-123. https://doi.org/10.1016/j.gene.2004.09.012

Likhith RK, Alagarasan G, Muthurajan R, Parasuraman B, Subramanian R (2021). Genome wide identification of mungbean (Vigna radiata [L.] R. Wilczek) Late Embryogenesis Abundant (LEA) protein gene family. Israel Journal of Plant Sciences 69(1-2):79-86. https://doi.org/10.1163/22238980-bja10049

Lin Y, She M, Zhao M, Yu H, Xiao W, Zhang Y, Li M, Chen Q, Zhang Y, Wang Y, He W (2024). Genome-wide analysis and functional validation reveal the role of late embryogenesis abundant genes in strawberry (Fragaria× Ananassa) fruit ripening. BMC Genomics 25(1):228. https://doi.org/10.1186/s12864-024-10085-9

Litts J, Erdman M, Huang N, Karrer E, Noueiry A, Quatrano R, Rodriguez R (1992). Nucleotide sequence of the rice (Oryzasativa) Em protein gene (Emp1). Plant Molecular Biology 19(2):335-337. https://doi.org/10.1007/BF00027357

Litts JC, Colwell GW, Chakerian RL, Quatrano RS (1987). The nucleotide sequence of a cDNA clone encoding the wheat Em protein. Nucleic Acids Research 15(8): 3607-3618. https://doi.org/10.1093/nar/15.8.3607

Liu Y, Zheng Y (2005) PM2, a group 3 LEA protein from soybean, and its 22-mer repeating region confer salt tolerance in Escherichia coli. Biochemical and Biophysical Research Communications 331(1):325-332. https://doi.org/10.1016/j.bbrc.2005.03.165

Luo D, Hou X, Zhang Y, Meng Y, Zhang H, Liu S, Wang X, Chen R (2019). CaDHN5, a dehydrin gene from pepper, plays an important role in salt and osmotic stress responses. International Journal of Molecular Sciences 20(8):1989. https://doi.org/10.3390/ijms20081989

Luo D, Zhang X, Li Y, Wu Y, Li P, Jia C, Bao Q, Zhou Q, Fu C, Liu W, Liu Z (2023). MsDIUP1 encoding a putative novel LEA protein positively modulates salt tolerance in alfalfa (Medicago sativa L.). Plant and Soil 487(1):547-66. https://doi.org/10.1007/s11104-023-05951-6

Ma J, Zuo D, Ye H, Yan Y, Li M, Zhao P (2023). Genome-wide identification, characterization, and expression pattern of the late embryogenesis abundant (LEA) gene family in Juglans regia and its wild relatives J. mandshurica. BMC Plant Biology 23(1):80. https://doi.org/10.1186/s12870-023-04096-z

Melgar AE, Rizzo AJ, Moyano L, Cenizo R, Palacios MB, Zelada AM (2024). Genome-wide identification and salt stress-expression analysis of the dehydrin gene family in Chenopodium quinoa. Current Plant Biology 38:100340. https://doi.org/10.1016/j.cpb.2024.100340

Min DH, Zhang XH, Xu ZS, Zhao Y, Chen Y, Li LC, Chen M, Ma YZ (2012). Induction kinetics of a novel stress-related LEA gene in wheat. Plant Molecular Biology Reporter 30(6):1313-1321. https://doi.org/10.1007/s11105-012-0446-2

Mota APZ, Oliveira TN, Vinson CC, Williams TCR, Costa MMdC, Araujo ACG, Danchin EG, Grossi-de-Sá MF, Guimaraes PM, Brasileiro ACM (2019). Contrasting effects of wild Arachis dehydrin under abiotic and biotic stresses. Frontiers in Plant Science 10:497. https://doi.org/10.3389/fpls.2019.00497

Mowla SB, Cuypers A, Driscoll SP, Kiddle G, Thomson J, Foyer CH, Theodoulou FL (2006). Yeast complementation reveals a role for an Arabidopsis thaliana late embryogenesis abundant (LEA)‐like protein in oxidative stress tolerance. The Plant Journal 48(5):743-756. https://doi.org/10.1111/j.1365-313X.2006.02911.x

Nair RM, Pandey AK, War AR, Hanumantharao B, Shwe T, Alam AK, Pratap A, Malik SR, Karimi R, Mbeyagala EK, Douglas CA (2019). Biotic and abiotic constraints in mungbean production—progress in genetic improvement. Frontiers in Plant Science 10:1340. https://doi.org/10.3389/fpls.2019.01340

Nawaz M, Sun J, Shabbir S, Khattak WA, Ren G, Nie X, Bo Y, Javed Q, Du D, Sonne C (2023). A review of plants strategies to resist biotic and abiotic environmental stressors. Science of the Total Environment 29:165832. https://doi.org/10.1016/j.scitotenv.2023.165832

Rajesh S, Manickam A (2006). Prediction of functions for two LEA proteins from mungbean. Bioinformation

(4):133. https://doi.org/10.6026/97320630001133

Raynal M, Gaubier P, Grellet F, Delseny M (1990). Nucleotide sequence of a radish cDNA clone coding for a late embryogenesis abundant (LEA) protein. Nucleic Acids Research 18(20):6132. https://doi.org/10.1093/nar/18.20.6132

Reddy PS, Reddy GM, Pandey P, Chandrasekhar K, Reddy MK (2012). Cloning and molecular characterization of a gene encoding late embryogenesis abundant protein from Pennisetum glaucum: protection against abiotic stresses. Molecular Biology Reports 39(6):7163-7174. https://doi.org/10.1007/s11033-012-1548-5

Rinne, PL, Kaikuranta PL, van der Plas LH, van der Schoot C (1999). Dehydrins in cold-acclimated apices of birch (Betula pubescens Ehrh.): production, localization and potential role in rescuing enzyme function during dehydration. Planta 209(4):377-388. https://doi.org/10.1007/s004250050740

Shi H, He X, Zhao Y, Lu S, Guo Z (2020). Constitutive expression of a group 3 LEA protein from Medicago falcata (MfLEA3) increases cold and drought tolerance in transgenic tobacco. Plant Cell Reports 39:851-860. https://doi.org/10.1007/s00299-020-02534-y

Shibuya T, Itai R, Maeda M, Kitashiba H, Isuzugawa K, Kato K, Kanayama Y (2020). Characterization of PcLEA14, a group 5 late embryogenesis abundant protein gene from pear (Pyrus communis). Plants 9(9):1138. https://doi.org/10.3390/plants9091138

Suzuki N, Rivero RM, Shulaev V, Blumwald E, Mittler R (2014). Abiotic and biotic stress combinations. New Phytologist 203(1):32-43. https://doi.org/10.1111/nph.12797

Swire-Clark GA, Marcotte WR (1999). The wheat LEA protein Em functions as an osmoprotective molecule in Saccharomyces cerevisiae. Plant Molecular Biology 39(1):117-128. https://doi.org/10.1023/A:1006106906345

Tan F, Sun N, Zhang L, Wu J, Xiao S, Tan Q, Uversky VN, Liu Y (2021). Functional characterization of an unknown soybean intrinsically disordered protein in vitro and in Escherichia coli. International Journal of Biological Macromolecules 166:538-49. https://doi.org/10.1016/j.ijbiomac.2020.10.211

Ukaji N, Kuwabara C, Takezawa D, Arakawa K, Fujikawa S (2001). Cold acclimation-induced WAP27 localized in endoplasmic reticulum in cortical parenchyma cells of mulberry tree was homologous to group 3 late-embryogenesis abundant proteins. Plant Physiology 126(4):1588-1597. https://doi.org/10.1104/pp.126.4.1588

Ulrich T, Wurtele E, Nikolau B (1990). Sequence of EMB-1, an mRNA accumulating specifically in embryos of carrot. Nucleic Acids Research 18(9):2826. https://doi.org/10.1093/nar/18.9.2826

Wang M, Li P, Li C, Pan Y, Jiang X, Zhu D, Zhao Q, Yu J (2014). SiLEA14, a novel atypical LEA protein, confers abiotic stress resistance in foxtail millet. BMC Plant Biology 14 (1):290. https://doi.org/10.1186/s12870-014-0290-7

Wang X, Liu H, Li Y, Zhang L, Wang B (2024). Heterologous overexpression of Tawzy1-2 gene encoding an SK3 dehydrin enhances multiple abiotic stress tolerance in Escherichia coli and Nicotiania benthamiana. Planta 259(2):39. https://doi.org/10.1007/s00425-023-04328-4

Wang Y, Chen G, Lei J, Cao B, Chen C (2020). Identification and characterization of a LEA-like gene, CaMF5, specifically expressed in the anthers of male-fertile Capsicum annuum. Horticultural Plant Journal 6(1):39-48. https://doi.org/10.1016/j.hpj.2019.07.004

Williams B, Tsang A (1991). A maize gene expressed during embryogenesis is abscisic acid-inducible and highly conserved. Plant Molecular Biology 16(5):919-923. https://doi.org/10.1007/BF00015086

Wise MJ, Tunnacliffe A (2004). POPP the question: what do LEA proteins do?. Trends in Plant Science 9(1):13-17. https://doi.org/10.1016/j.tplants.2003.10.012

Wu C, Hu W, Yan Y, Tie W, Ding Z, Guo J, He G (2018). The late embryogenesis abundant protein family in cassava (Manihot esculenta Crantz): Genome-wide characterization and expression during abiotic stress. Molecules 23(5):1196. https://doi.org/10.3390/molecules23051196

Xu D, Duan X, Wang B, Hong B, Ho THD, Wu R (1996). Expression of a late embryogenesis abundant protein gene, HVA1, from barley confers tolerance to water deficit and salt stress in transgenic rice. Plant physiology 110(1):249-257. https://doi.org/10.1104/pp.110.1.249

Yamada A, Saitoh T, Mimura T, Ozeki Y (2002). Expression of mangrove allene oxide cyclase enhances salt tolerance in Escherichia coli, yeast, and tobacco cells. Plant and Cell Physiology 43(8):903-910. https://doi.org/10.1093/pcp/pcf108

Yang J, Kim HE, Jung YH, Kim J, Kim DH, Walmsley AR, Kim KH (2020). Zmo0994, a novel LEA-like protein from Zymomonas mobilis, increases multi-abiotic stress tolerance in Escherichia coli. Biotechnology for Biofuels 13:1-5. https://doi.org/10.1186/s13068-020-01790-0

You FM, Huo N, Gu YQ, Luo MC, Ma Y, Hane D, Lazo GR, Dvorak J, Anderson OD (2008). BatchPrimer3: a high throughput web application for PCR and sequencing primer design. BMC Bioinformatics 9:1-13. https://doi.org/10.1186/1471-2105-9-253

Yu JN, Zhang JS, Shan L, Chen SY (2005). Two new group 3 LEA genes of wheat and their functional analysis in yeast. Journal of Integrative Plant Biology 47(11):1372-1381. https://doi.org/10.1111/j.1744-7909.2005.00126.x

Zan T, Li L, Li J, Zhang L, Li X (2020). Genome-wide identification and characterization of late embryogenesis abundant protein-encoding gene family in wheat: evolution and expression profiles during development and stress. Gene 736:144422. https://doi.org/10.1016/j.gene.2020.144422

Zeng X, Ling H, Yang J, Li Y, Guo S (2018). LEA proteins from Gastrodia elata enhance tolerance to low temperature stress in Escherichia coli. Gene 646:136-142. https://doi.org/10.1016/j.gene.2018.01.002

Zhang L, Ohta A, Takagi M, Imai R (2000). Expression of plant group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed functional divergence among LEA proteins. The Journal of Biochemistry 127(4):611-616. https://doi.org/10.1093/oxfordjournals.jbchem.a022648

Zhang YM, Wang HQ, Liu DM, Liu RJ (2020). Three tandemly aligned LEA genes from Medicago truncatula confer differential protection to Escherichia coli against abiotic stresses. Biologia Plantarum 64(1). https://doi.org/10.32615/bp.2019.112

Zhao W, Yao F, Zhang M, Jing T, Zhang S, Hou L, Zou Z (2016). The potential roles of the G1LEA and G3LEA proteins in early embryo development and in response to low temperature and high salinity in Artemia sinica. PLoS One 11 (9):e0162272. https://doi.org/10.1371/journal.pone.0162272

Downloads

Published

2024-09-06

How to Cite

SUBRAMANIAN, R., PANDI, N. U., THANGAVEL, R., SWAMY, L. R., LAKSHMINARAYANAN, S. P., SANTHAMANI, S., M. RAVALAN, B., & JACOB, R. (2024). Functional validation of mungbean LEA protein coding gene in bacterial expression system confers salt stress tolerance. Notulae Botanicae Horti Agrobotanici Cluj-Napoca, 52(3), 13416. https://doi.org/10.15835/nbha52313416

Issue

Section

Research Articles
CITATION
DOI: 10.15835/nbha52313416